| Background and Objective:Staphylococcus aureus (S.aureus) is one of the important pathogens of nosocomial and community-acquired infections. Methicillin-resistant staphyloco-ccus aureus (MRSA) was multidrug-resistant and became famous super bacteria. MRSA can cause a variety of progressive and necrotic diseases, such as bacteremia, toxic shock syndrome, hematosepsis and so on. At the same time, antibiotic resistance of MRSA isolates was severity and showed multidrug resistance status, especially the emergence of vancomycin intermediate or resistant strains in recent years. Therefore, monitoring of MRSA epidemic and drug resistance has become a major public health concern worldwide. It was noteworthy that China was one of the most severe countries of MRSA epidemic, and MRSA, hepatitis B and AIDS were called the "three refractory infection diseases" that are threatening the world.Previous studies on MRSA confined mainly to the relation between hospital- and community-associated MRSA. The latest research shows that the epidemiological pattern of MRSA has changed. MRSA can transmit among animal food, animal and animal practitioners, called livestock-associated MRSA. Worryingly, it has been reported that LA-MRSA can invade healthy people without animal contact history, and even invade into hospitals and cause nosocomial infections. It suggests that LA-MRSA may cross species to spread to healthy people and patients in the hospital, so it will cause a great threat to human health and life.It is still unclear about the route of cross-species transmission of LA-MRSA that cause human infections. Research in foreign countries mainly confined to MRSA monitoring study on livestock, breeders and veterinary in farms, and research in China mainly confined to MRSA colonization in various livestock and pets. In China, it was still lack of detailed study of MRSA colonization and molecular characteristics of human with animal contact, and was also lack of detailed reports on the influencing factors of MRSA colonization. There still have no reports in the literature that study the molecular characteristics and cross transmission of MRSA among animal practitioners, animals and environment at the same time.This study intends to carry out molecular epidemiological study on animal (including pigs, cattle, sheep, and poultry), animal practitioners and general community residents (refered to controls) in animal farms, animal slaughtering places and places of sale. The homology between animal and human MRSA was analyzed, so as to clear LA-MRSA cross-species transmission from animal to human. This study will explore new ideas for revealing the mechanism of LA-MRSA cross-species transmission, and has important scientific significance in controlling of LA-MRSA epidemic and transmission among human.Methods:This cross-sectional study was conducted in Guangdong province, China, between November 2013 and November 2014. A multistage sample design was used to obtain workers with occupational livestock contact and controls. We carried out nasal swab sampling and questionnaire survey of animal practitioners and controls. The questionnaire survey includes:basic demographic characteristics, self-reported occupational livestock contact, livestock contact in living space, disease history and medication history in the last year, living habits, family members in the medical and health system and so on. We also carried out cotton swab sampling of environment and nasal swab sampling of animal in farms, meat retail markets and slaughterhouses.Initial identification of S. aureus was based on traditional biochemical tests, and then confirmed by PCR screening for the carriage of 16S rRNA and nuc genes. S. aureus isolates that were positive for oxacillin resistance or the mecA gene were identified as MRS A. Antimicrobial susceptibility for isolates identified as S. aureus were performed with antimicrobial susceptibility test, including penicillin, oxacillin, cefoxitin, gentamycin sulfate, erythromycin, tetracycline, ciprofloxacin, levofloxacin, furadantin, paediatric compound sulfamethoxazole, clindamycin, chloromycetin, rifampin, linezolid and vancomycin. The interpretations of results were based on the Clinical and Laboratory Standards Institute guidelines 2012. Isolates that demonstrated complete resistance to three or more classes of antibiotics were classified as multi-drug resistant strains of S. aureus (MDRSA).All MRSA isolates were characterized by SCCmec genotyping using multiple PCR. Seven housekeeping genes of S. aureus isolates were amplified by the PCR method, and the products of PCR amplification were purified by kits and then sequenced. The sequencing results were submitted to the MLST website (http://www. mlst.net/), and alleles and STs were assigned using the MLST database. CCs were determined using eBURST version 3 (http://eburst.mlst.net) and the stringent group definition (6/7 shared alleles).All data were entered in duplicate into the EpiData version 3.1 database, and consistency test was used to automatically detect data entry errors. All statistical analyses were conducted using STATA version 13.0 (StataCorp LP, College Station, Texas, USA). The chi-square test and Fisher exact test were used to compare rates, and univariable and multivariable non-conditional logistic regression models were used to analyze the influencing factors. The trend relations between occupational livestock contact (including frequency, short-term duration, long-term duration) and human MRSA carriage were examined using logistic regression models. We defined a two-sided p-value of ≤0.05 as being of statistical significance.BURST (Based upon related sequence types) algorithm was used to compare the evolutionary relation of all alleles. The UPGMA was used to analyze the correlation between ST types. The phylogenetic tree (UPGMA tree) was constructed according to the ST allele mismatch matrix.Results:A total of 1178 controls without occupational animal contact were effectively interviewed, and the overall prevalence of MRS A was 1.4%(16/1178). These MRS A isolates were mainly resistant to beta-lactam antibiotics (100%), clindamycin (68.8%), erythromycin (62.5%), and tetracycline (50%). About 81.3%(13/16) MRSA was in multi-drug resistance, and the main resistant pattern was resistant to β-lactam antibiotics, clindamycin and erythromycin at the same time. The ST types of MRSA isolates from controls were mainly ST7 (43.8%) and ST59 (12.5%), and the SCCmec genotyping was mainly community-associated SCCmec-IVand SCCmec-V. None of 109 MRSA isolates from controls was positive for pvl gene.A total of 682 animal practitioners were effectively interviewed, and the overall prevalence of MRSA was 7% (48/682), including 10.7% (26/244) for breeders and veterinarian, and 5% (22/438) for slaughterhouse workers and butchers. These MRSA isolates were mainly resistant to beta-lactam antibiotics (100%), clindamycin (83.3%), erythromycin (81.3%), and tetracycline (72.9%). About 87.5% MRSA was in multi-drug resistance, and the main resistant pattern was resistant to P-lactam antibiotics, clindamycin, erythromycin and tetracycline at the same time. The rates of antibiotics-resistance and multi-drug resistance of MRSA isolates from animal practitioners were much more serious than that in controls. The ST types of MRSA isolates from animal practitioners were mainly ST9 (33.3%), ST59 (12.1%), ST7 (10.4%) and ST45 (10.4%), and the SCCmec genotyping was mainly community-associated SCCmec-IVand SCCmec-V. Only one MRSA-ST59 isolate was positive for pvl gene.A total of 310 pigs were effectively sampled, and the overall prevalence of MRSA was 2.3% (7/310). These MRSA isolates from pigs were all resistant to clindamycin (100%), erythromycin (100%) and tetracycline (100%). All pig MRSA were in multi-drug resistance, and the main pattern was resistant to β-lactam antibiotics, clindamycin, erythromycin and tetracycline at the same time. The ST types of MRSA isolates from pigs were mainly ST9 (6 isolates) and ST6 (1 isolate), and the SCCmec genotyping was mainly community-associated SCCmec-Ⅳ. None of 7 MRSA isolates from pigs was positive for pvl gene.A total of 299 environmental samples were collected, and the overall prevalence of MRSA was 4.3% (13/299). Thirteen MRSA isolates from environmental samples were maily resistant to β-lactam antibiotics (100%), tetracycline (76.9%) and cSMZ-TMP (76.9%). About 53.8% MRSA isolates were resistant to clindamycin, erythromycin and SMZ-TMP. About 76.9% MRSA isolates were in multi-drug resistance, and the main resistant pattern was resistant to β-lactam antibiotics, clindamycin, erythromycin and tetracycline at the same time. The ST types of 13 MRSA isolates from farms, slaughterhouses, meat markets environment were diversity, and mainly belonged to ST7(5 isolates) and ST59(2 isolates). The SCCmec genotyping was mainly community-associated SCCmec-IVand SCCmec-V. None of 13 MRSA isolates was positive for pvl gene.When compare the ST typing of S. aureus among animals, animal practitioners and controls, we found:animals, animal practitioners and controls carried the same human-associated ST59 and ST6 S.aureus isolates, suggesting that these isolates may cross-species transmit from human to animals, and cause animal colonization and infections. The ST9 S.aureus isolates found only in animal and practitioners, and controls did not carry these animal-associated ST9 S.aureus isolates which were popular in Asian. This will suggest that these ST9 isolates may cross-species transmit from animals to human, and cause human colonization and infections. To note, we found when compare the ST typing of MRSA isolates from animals and human:the ST9 MRSA isolates found only in animal and practitioners, and controls did not carry these animal-associated ST9 MRSA isolates which were popular in Asian. These results suggest that ST9 MRSA isolates may cross-species transmit from animals to human, and cause human LA-MRSA colonization and infections.When compare the ST typing ofS. aureus among environment and human isolates, we found:samples from environment, animal practitioners and controls were tested the same human-associated ST7, ST59, ST88, ST1, ST5, ST45 and ST2359 S.aureus isolates, suggesting that these isolates may cross-species transmit between human and environment. To note, we found when compare the ST typing of MRSA isolates from environment and human:ST2359 MRSA (belong to CC9) found only in environment and animal practitioners, but controls did not carry these animal-associated ST2359 MRSA which was popular in Asian. These results suggest that ST2359 MRSA may transmit from animals or environment to human, and cause human MRSA colonization and infections. The human-associated ST7 MRSA and ST59 MRSA isolates were found in environment, animal practitioners and controls, suggesting that these isolates may transmit between human and environment.When analyze the relation between occupational animal contact and human MRSA carriage, the results showed that:compared with no contact, workers with occupational contact with any types of livestock experienced a significantly higher risk of MRSA carriage (AOR=6.27,95%CI 3.42-11.50). When examining the relation by types of livestock, similar positive association was observed among workers with occupational contact with pigs (AOR=6.50,95%CI 3.47-12.19) or poultry (AOR=4.92,95%CI 1.33-18.27). But no significant associations were found in workers with occupational contact with cows or sheep, possible due to small numbers.When analyze the trend relation between frequency of animal contact and human MRSA carriage, the results showed that:The risk for being MRSA carrier increased with increasing frequency of occupational livestock contact (AOR=2.25,95%CI 1.72-2.94). When examining the frequency-risk relation by types of livestock, similar positive trend association was observed among workers with occupational contact with pigs (that is, the risk for being MRSA carrier increased with increasing frequency of occupational pig contact). When analyze the trend relation between duration of occupational animal contact and human MRSA carriage, the results showed that:Short-term duration of occupational livestock contact was associated with increased risk of MRSA carriage in a monotonically increasing manner(AOR=1.58,95%CI 1.29-1.95). Long-term duration-risk increased trend was also observed for occupational livestock contact(AOR=1.25,95%CI 1.16-1.34). When examining the duration-risk relation by types of livestock, similar duration-risk relations were observed among workers with short-term and long-term occupational contact with pigs.It is notable that previous studies have revealed the positive relation between occupational livestock contact and human MRS A carriage, but it is still unclear the relation between occupational livestock contact and human carriage of multi-drug resistant S.aureus. Our study indicated:Workers with occupational contact with any types of livestock experienced a significantly higher risk of MDRSA carriage (AOR=2.91,95% CI 1.87-4.52), and workers with occupational pig contact also experienced a significantly higher risk of MDRSA carriage (AOR=2.89,95% CI 1.82-4.58). Occupational livestock contact can significantly increase the risk of human MDR-MRSA carriage (AOR=7.20,95% CI 3.69-14.05), and occupational pig contact can also significantly increase the risk of human MDR-MRSA carriage (AOR=7.84,95% CI 3.94-15.61). Thus, there are strong positive correlation between occupational livestock contact and multi-drug resistant isolates. The risk for being MDRSA and MDR-MRSA carrier increased with increasing frequency of occupational livestock contact, and there is an obvious monotonically increasing trend. Short-term duration of occupational livestock contact was associated with increased risk of MDR-MRSA carriage in a monotonically increasing manner. However, long-term duration-risk trend was also observed for occupational livestock contact and human carriage of MDRSA and MDR-MRSA isolates.There were no significant relations between hospital-associated factors (such as previous antimicrobial use history, outpatient history, hospitalization history, operation history, skin damage/inflammation history, and having family members of medical staff) and human MRSA carriage. There were also no significant relations between community-associated factors (such as smoking, drinking, drinking tea, washing hands or taking a shower as soon as return home after work, and having livestock in living places) and human MRSA carriage.Conclusions:(1) Occupational livestock contact may significantly increase the risk of human MRSA carriage. There is a monotonically increasing trend between frequency of occupational livestock contact and human MRSA carriage. There are also monotonically increasing relations between short-term or long-term duration of occupational livestock contact and human MRSA carriage. In conclusion, occupational livestock contact is the key fator of human MRSA carriage.(2) The S.aureus and MRSA strains isolated from pigs are in multi-drug resistance, and the antibiotics-resistant status can not be ignored. The antibiotics-resistant patterns of MRSA isolates from animal, human and environment was consistent, and were mainly resistant to β-lactam antibiotics, clindamycin, erythromycin and tetracycline at the same time.(3) The ST9 MRSA isolates found only in animal and practitioners, and controls did not carry livestock-associated ST9 MRSA which was popular in Asian. These results suggest that ST9 MRSA may cross-species transmit from livestock to human, and cause human LA-MRSA colonization and infections. The ST2359 MRSA isolates (belong to CC9) found only in environment and practitioners, and controls did not carry these animal-associated ST2359 MRSA isolates which were popular in Asian. These results suggest that ST2359 MRSA isolates may transmit from animals or environment to human, and cause human MRSA colonization and infections.(4) The human-associated ST7 MRSA and ST59 MRSA isolates were found in environment, animal practitioners and controls, suggesting that these isolates may transmit between human and environment. |
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